It has been only 10 years since the first reports that macrophages infiltrate adipose tissue (AT) in obesity and contribute to insulin resistance (IR). Since then, new advances in the field have included demonstration that 1) macrophages form crown-like structures around dead adipocytes, 2) other leukocytes such as T lymphocytes, B cells, mast cells, and neutrophils are also recruited to AT, and 3) the AT macrophages can be polarized toward an M1 or M2 phenotype. To date, the polarization of ATMs has been used as an indication of whether they are resident and anti-inflammatory (M2) or recruited and inflammatory (M1). The prevailing model of ATM polarization holds that 'resident' ATMs display an 'alternatively activated' M2 phenotype, whereas macrophages recruited to adipose tissue during the onset of obesity exhibit a predominantly M1 'classical activation' state. The M2 macrophages are thought to play a role in tissue remodeling and repair in other organs. However, the physiologic function of M2 macrophages in AT is not known. In this application, we propose a role for M2 AT macrophages in local iron recycling and homeostasis, thus providing a specific function for these cells within the AT microenvironment. Furthermore, we propose that obesity reduces the ability of M2 macrophages to handle iron properly, thus contributing to adipose tissue dysfunction. In other organs, liver and spleen in particular, macrophages play a key role in iron homeostasis. Both M1 and M2 macrophages are known to sequester iron. The role of M1 iron sequestration is in bacteriostasis; however, the role of M2 iron sequestration is unknown. We have exciting preliminary data demonstrating that about 5% of AT macrophages are paramagnetic and sequester iron. These macrophages have gene expression patterns reflective of an M2 polarization with elevated expression of iron metabolism-related genes such as hemoxygenase-1, ferroportin, transferrin receptor, and the ferritin polypeptides. Hereafter, we refer to these cells as MFehi cells. The MFehi cells are present in the AT of both low fat (LF) and high fat (HF) fed mice. On a cellular basis, the MFehi cells contain less iron than the MFelo cells and their gene expression profiles indicate that they are defective in iron recycling compared to MFehi cells from the LF diet-fed mice. The overall goal of this Exploratory/Discovery R21 application is to characterize these AT MFehi cells and to determine their contribution to AT dysfunction in obesity. We will take an unbiased approach to characterizing the MFehi cells and will evaluate how they interact with their surrounding adipocytes and MFelo cells. PUBLIC HEALTH RELEVANCE: Obesity has become a worldwide epidemic resulting in increased incidence of many different diseases. Expanded and inflammatory adipose tissue is likely one of the primary contributors to the pathological consequences of obesity. Identification and characterization of how iron handling in these adipose tissue macrophage influences adipose tissue expansion is important for understanding the metabolic consequences of obesity.